The long-term objectives of this research proposal are to elucidate the mechanism(s) of transcription and replication of vesicular stomatitis virus (VSV), a prototypic nonsegmented, negative-strand RNA virus. VSV has served as an excellent paradigm for members of this group of viruses that include some of the most serious human pathogens such as rabies, respiratory syncytial, measles, and parainfluenza viruses. Deciphering the basic principles that govern faithful and efficient expression of the viral genome will provide significant insights into the viral life cycle. Knowledge of these basic principles is important for rational design of antiviral therapeutics and for generating live attenuated viruses for vaccines. This proposal outlines several lines of investigation to test a number of ideas and hypotheses for a broader understanding of key cis-acting regulatory signals in the viral genome and a trans-acting protein factor encoded in the genome that play important roles in the genetic expression of VS V. A well-defined reverse genetic system that is entirely dependent on viral components derived only from transfected plasmids will be used to address the role of specific regions within the viral genome in transcription and replication. Mutations in the viral genome will be introduced and the effects of these mutations on transcription and replication activities will be examined. The P protein, an integral component of the viral RNA polymerase will be examined for its functions and specific interactions with the cis-acting sequence elements in the viral genome and other trans-acting protein factors. The specific aims of this proposal are to: (i) investigate the transcription regulatory signals and understand the mechanism of transcription; (ii) delineate encapsidation, replication, and packaging signals in the viral genome; and (iii) examine how interactions of the P protein with the RNA synthetic machinery regulate viral genome transcription and replication. These studies are of major importance for an understanding of the mechanisms of the viral gene expression and virus growth.